Screening And Identification Of PRKAA1 Gene-related LncRNA And Its Expression Regulation In Intramuscular Fat

PRKAA1,a member of AMPK family,is an important protein kinase in organisms and plays an important regulatory role in energy homeostasis,fat metabolism and cell proliferation.LncRNA plays an important role in embryo development,fat deposition and cell differentiation.In Guizhou Congjiang xiang pig and large white pig as the research material,through the qRT-PCR,molecular cloning,cell culture,cell transfection,Western blot,screening and identification of relevant PRKAA1 LncRNA-ALNC1 and ALNC2 gene,detect fat related gene expression level,analyze its in intramuscular,subcutaneous fat precursor cells,and regulatory mechanism of adipocyte differentiation,discussing local pig meat quality traits,parsing LncRNA influence on pig fat deposition and meat quality traits,to understand local pig meat quality traits in China,Excavation,conservation and innovative utilization of local pig resources have important scientific significance and potential application prospects.1.Screening and identification of porcine PRKAA1 gene-related LncRNAs and their expression analysis in tissuesThe online software NCBI and NONCODE(http://www.noncode.org/)were used to screen and identify PRKAA1-related LncRNA.Results The PRKAA1gene-related LncRNAs(NONSUST030689.1,ALNC1)and(NONSUST004358.1,ALNC2)were successfully screened.Sequence analysis showed that ALNC1 is 935 bp in length and is located on chromosome 17 and ALNC2 is 1639 bp in length and is located on chromosome 11.The cloned sequence is inserted 46 bp more than the original sequence.Both have a rich secondary structure and multiple open reading frames.It has a target of action with PRKAA1 and interacts with miR-1226-5p,miR-487b-5p,miR-7156-5p and so on.Tissue qRT-PCR results showed that ALNC1 was the highest expressed in the liver and large intestine of Cong Jiang xiang pig and large white pig,and the expression of fat and longest muscle tissue in Cong Jiang xiang pig was significantly higher than that of large white pig(P < 0.01),ALNC2 expression was highest in the spleen and lungs of Cong Jiang xiang pig and large white pig,and the expression of fat from Cong Jiang xiang pig was significantly lower than that of large white pig(P <0.05),and the expression of longest back muscle was significantly higher than that large white pig(P <0.01).It is speculated that two LncRNAs may be important candidate genes affecting pig fat deposition.2.Expression analysis of ALNC1 and ALNC2 during the induction and differentiation of primary fat precursor cellsFive-day-old primary intramuscular and subcutaneous fat precursor cells from Cong Jiang xiang pig were isolated and cultured.The qRT-PCR method was used to detect the expression of two LncRNA and PRKAA1 cells at time points of 0 h,24 h,36 h,48 h,60 h,72 h,96 h,and 120 h.The results showed that the expression trends of PRKAA1 and ALNC1 genes in intramuscular fat precursor cells were the same,rising first and then decreasing,and the highest expression was at 60 h;ALNC2 was the highest expression at 72 h;in subcutaneous fat precursor cells,the PRKAA1 gene expression trend was the same as Intramuscular precursor cells were the same,with the highest expression at 48 h;ALNC1 and ALNC2 genes showed the same expression trend,both of which decreased first,then increased and then decreased,and the expression was highest at 72 h.The expression patterns of PRKAA1,ALNC1,and ALNC2 and fat differentiation-related genes PPARγ,FAS,ACC,and ATGL during the differentiation of primary adipose precursor cells from Cong Jiang xiang pig are basically the same,indicating that ALNC1 and ALNC2 are involved in the regulation of adipose precursor cell differentiation.3.ALNC1 and ALNC2 function in the differentiation of precursor adipocytesConstruction of eukaryotic expression vectors pEGFP-ALNC1 and pEGFP-ALNC2,and synthesis of siRNA against ALNC1.After transfection of adipose precursor cells,qRT-PCR and Western blotting were used to detect fatmetabolism-related genes PRKAA1,PPARγ,FAS,and HSL in mRNA and Expression of protein levels,and triglyceride and cholesterol levels in adipocytes were determined simultaneously.The results showed that after over-expression of ALNC1,the mRNA and protein levels of PRKAA1 and FAS were up-regulated and the expression of PPARγ protein was down-regulated in intramuscular fat precursor cells.The expression of PRKAA1,PPARγ mRNA and protein were up-regulated in subcutaneous fat precursor cells,and HSL and FAS were up-regulated.mRNA expression was down-regulated;after interfering with ALNC1,PRKAA1,FAS,and HSL mRNA and protein levels were down-regulated in intramuscular fat precursor cells,and PPARγ mRNA expression was up-regulated;PRKAA1 and HSL mRNA and protein expression were down-regulated in subcutaneous fat precursor cells,and PPARγ protein expression was up-regulated.After overexpression of ALNC2,PRKAA1,FAS,and PPARγ mRNA and protein levels were up-regulated in intramuscular fat precursor cells,and HSL protein expression was down-regulated;PRKAA1 and HSL mRNA and protein levels were up-regulated in subcutaneous fat precursor cells,FAS mRNA levels were down-regulated,and PPARγ Protein levels are down-regulated.Overexpression of ALNC1,ALNC2,and interference with ALNC1 increased and decreased triglyceride and cholesterol levels in the two precursor adipocytes,respectively.According to the obtained results,this paper filter to get two pigs PRKAA1 gene related LncRNA ALNC1 and ALNC2,and two LncRNA in Congjiang xiang pig and large white pig common expression in different tissue,The cloned sequence of ALNC2 from Jiangxiang pig liver was inserted 46 bp from the original sequence,which may be used as a molecular marker fragment for breeding from Jiangxiang pig.The expression patterns of PRKAA1,ALNC1 and ALNC2 and adipogene-related genes were basically the same in the differentiation process of primary adipogenitor cells.In porcine adipose precursor cells,ALNC1 and ALNC2 have regulatory effects on cell differentiation related genes PRKAA1,FAS,PPARγand HSL and promote the differentiation of adipose precursor cells and the formation of fat.